Fig 1: Sepiapterin promotes MUC2 and Cytokeratin-20 expression in Caco-2:HT-29 epithelial layer and the emergence of M1-like phenotype in naïve THP-1 macrophages in 3D Flipwell co-cultures. (A) (Left) Confocal images of Caco-2:HT-29 cells co-cultured with THP-1 cells in the 3D Flipwells and stained for CK20 (green) and MUC2 (red). Cytokeratin staining is very pronounced in the areas surrounding mucus production as indicated by MUC2 expression. Scale bar represents 50 µm. The original raw images are included in Supplementary Information file. (Right) Percent positive cells for CK20 and MUC2. (B) (Left) Confocal images of THP-1 cells cocultured with Caco-2:HT-29 in the 3D Flipwells and stained for Pan Macrophage marker CD68 (red) and CD80 (green). Pan Macrophage marker CD68 is present in both SEP treated and untreated control samples. CD80, typical of the M1 macrophage phenotype, is more pronounced in SEP treated sample with long pseudopods extending outward typical of the M1 phenotype. (Right) Mean intensities of CD68 and CD80. (C) (Left) Confocal images of THP-1 cells cocultured with Caco-2:HT-29 in the 3D Flipwell and stained for Pan Macrophage marker CD80 (green) and CD163 (red) to validate phenotypic polarization toward M1 phenotype and away from M2 phenotype. Scale bar represents 25 µm. The original raw images are included in Supplementary Information file. (Right) Mean intensity of CD80 and CD163. Error bars: mean ± STDEV. *p < 0.05; ***p < 0.001.